Project Summary The objective of this exploratory research is to create a high-fidelity tool to analyze cortical tension and cell-cell adhesion in their co-active states. These properties and their coupling play ubiquitous roles in the organization of cell aggregates, tissues, and cell clusters, in biological processes such as morphogenesis, cancer metastasis, and wound healing. They are the key cellular-level properties that affect and control the collective multi-cellular behavior, especially in regimes where strong bonds between cells and the extracellular matrix are absent or have not formed. Experimental and systematic quantification of these properties is nonetheless challenging, and more so than simply acquiring the apparent moduli of the cell cortex. In particular, both prior findings and our preliminary results corroborate that the current techniques used to quantify cortical tension may have resulted in its appreciable alteration due to the interrogative force applied, and hence the acquired value does not reflect that in the ?true and nave? state. This proposal is developed to address this challenge. Our rationale derives from a state-of-the-art understanding in single cell mechanics. We hypothesize that minimal impact to and faithful quantification of the cell mechanical states can not be achieved unless one avoids the so-called soft glassy rheology (?fluidized?) regime. This requires us to focus on the high frequencies or short timescales, known as the worm-like chain (WLC) regime. Collective knowledge in the field suggests that in this regime we may be able to interrogate the cell cortex without appreciably modifying it. Our approach is formulated with two innovations: 1) A rapid deformation-relaxation analysis, combined with 2) simultaneous mechanical and fluorescence quantification of cell couplets. Our aims are: Aim I To establish mechanical analysis via rapid electrodeformation-relaxation. We will develop the necessary experimental and analytical tools for such purpose, and validate our main hypothesis. Aim II To interrogate tension-adhesion coupling by studying couplets. A couplet is a pair of cells bound to each other via adhesion, and is the ideal, minimal unit in which we can observe the co-action of tension and adhesion. We will combine the mechanical analysis in Aim 1 with fluorescence imaging of both de novo and mature couplet cells stained for actin and N-cadherin observation. Both adhesion and tension will be modulated and their effects quantified. The success of this project will provide an innovative and convenient tool, and generate the much needed data on tension-adhesion coupling, for model building, mechanism-based understanding, and hypothesis validation, in broad fields where cell-cell mechanical interactions play essential roles.